This invention relates to treatment of human papilloma virus (HPV) infection.
Papilloma viruses are non-enveloped DNA viruses with a double stranded circular genome of approximately 8,000 bp. Over 75 types of human papilloma viruses (HPV) have been typed at the DNA level, and these can be broadly grouped into families on the basis of their tissue tropism.
Histologic, molecular, and epidemiologic evidence have implicated some HPV strains in cervical dysplasia and cervical cancer. Many studies support the view that most moderate and severe cervical intraepithelial neoplasias (CIN) contain HPV DNA which is exclusively detected in the histologically abnormal epithelium of these lesions. Persistent infection with HPV is believed to be the predominant risk factor for development of cervical carcinoma. HPV DNA is readily found in episomal form within cells exhibiting a cytopathic effect, while the HPV DNA is found integrated within the chromosomes of cells associated with most high grade precancerous lesions and cancer. Approximately 23 HPV types are commonly found in anogenital screening programs, but only 10-15 are associated with progressive disease. Type 16 is the type most commonly found in cervical cancer tissue.
Papillomaviruses contain nine open reading frames. HPV genes with transforming properties have been mapped to open reading frames E6 and E7. Substantial biochemical work has demonstrated that the HPV E6 protein inactivates the protein p53, whereas the E7 protein interferes with retinoblastoma (Rb) protein function. Since p53 and Rb are tumor-suppressor proteins which function as cell division inhibitors, their inactivation by E6 and E7 leads the cell to enter into S phase of the cell cycle. Expression of E6 and E7 is sufficient to immortalize some primary cell lines, and blocking E6 or E7 function has been shown to reverse the transformed state.
The invention is based on the discovery that a 13 amino acid peptide from the HPV strain 16 E7 protein that contains overlapping class I HLA binding, T cell epitopes can induce a CTL response in an animal. Accordingly, the invention includes an immunogenic peptide having within its sequence multiple class I MHC-binding epitopes from a human papillomavirus (HPV) protein, and which has a length of less than 19 amino acids and includes the sequence of Leu Met Gly Thr Leu Gly Ile Val Cys Pro Ile Cys (SEQ ID NO:16) (hereinafter xe2x80x9cimmunogenic peptidexe2x80x9d). The immunogenic peptide can optionally include sequences in addition to those derived from the E7 protein.
The immunogenic peptide can have the sequence of Leu Leu Met Gly Thr Leu Gly Ile Val Cys Pro Ile Cys (SEQ ID NO:3) or Xaa Leu Met Gly Thr Leu Gly Ile Val Cys Pro Ile Cys, Xaa being Met, Ala, Ser, Arg, Lys, Gly, Gln, Asp, or Glu (SEQ ID NO:19), e.g., Ala Leu Met Gly Thr Leu Gly Ile Val Cys Pro Ile Cys (SEQ ID NO:4).
The invention also includes the peptides Thr Leu Gly Ile Val Cys Pro Ile (SEQ ID NO:20) and Gly Thr Leu Gly Ile Val Cys Pro Ile (SEQ ID NO:21), as well as Xaa Thr Leu Gly Ile Val Cys Pro Ile (SEQ ID NO:27) and Gly Thr Leu Gly Leu Gly Ile Val Cys Pro Ile (SEQ ID NO:28), Xaa being Met, Ala, Ser, Arg, Lys, Gly, Gln, Asp, or Glu.
In addition, all of the peptides discussed herein may include additional amino acids to facilitate expression, e.g., an amino terminal methionine to facilitate translation.
The invention also includes a polypeptide having the sequence of a first peptide linked to a second peptide by a peptide bond. The first peptide (which can be at the carboxy terminus or the amino terminus of the second peptide, so long as it functions in that site) is a peptide which controls intracellular trafficking of a peptide to which it is attached, and the second peptide is the immunogenic peptide described above. The polypeptide may optionally be modified to introduce an amino acid substitution at the junction between the first and second peptides to promote cleavage of the first and second peptides by a signal peptidase.
The trafficking peptides can be any recognized signal sequence, e.g. a signal sequence from the adenovirus E3 protein. A preferred trafficking peptide is the signal peptide of HLA-DRxcex1, Met Ala Ile Ser Gly Val Pro Val Leu Gly Phe Phe Ile Ile Ala Val Leu Met Ser Ala Gln Glu Ser Trp Ala (SEQ ID NO:18).
The invention in addition includes a therapeutic composition containing the immunogenic peptide described above, and a pharmaceutically acceptable carrier. The polypeptide can optionally be formulated in a microparticle, a liposome or an immune-stimulating complex (ISCOM) (which may contain saponin alone as the active ingredient), or any other vehicle suitable for delivering into subjects the immunogenic peptides of the invention. When a microparticle is used, it preferably has a polymeric matrix that is a copolymer such as poly-lactic-co-glycolic acid (PLGA).
An immune response (e.g., a cellular immune response, including an MHC class I-mediated or class II-mediated immune response) in a mammal can be elicited by administering the immunogenic peptide to a mammal, e.g., a human, non-human primate, dog, cat, rabbit, cow, mouse, rat, guinea pig, or hamster, that has an MHC molecule that binds to the immunogenic peptide. The immunogenic peptide can be administered as part of a microparticle, liposome, or ISCOM, or in solution.
Another way to administer the peptide utilizes a nucleic acid, e.g., an expression vector, comprising a coding sequence encoding the immunogenic peptide. The nucleic acid can optionally encode a signal sequence linked to the immunogenic peptide, as described above. When the nucleic acid encodes such a signal sequence, it is preferred that it encodes the signal sequence from HLA-DRxcex1 (SEQ ID NO:18). In such a case, the immunogenic peptide can have the sequence, for example, of SEQ ID NO:4 or SEQ ID NO:3. Preferably, the nucleic acid does not include sequences from a viral genome that would render the nucleic acid infectious, and does not encode an intact E7 protein.
The nucleic acid described above can be included in a plasmid, optionally provided in a microparticle that also includes a polymeric matrix. In preferred embodiments, the polymeric matrix consists essentially of a copolymer of PLGA. The microparticle preferably has a diameter of, e.g., 0.02 to 20 microns, or less than about 11 microns. A plurality of microparticles preferably has diameter of, e.g., 0.02 to 20 microns, or less than about 11 microns
Also within the invention is a cell containing the plasmid of the invention. The cell can, e.g., be a B cell or other antigen presenting cell (APC). The cell may be cultured or otherwise maintained under conditions permitting expression of the peptide from the plasmid encoding it.
The nucleic acid and plasmid of the invention are useful in a method of inducing an immune response in a mammal, e.g., a human, by administering the above-described plasmid to the mammal, e.g., as xe2x80x9cnaked DNAxe2x80x9d. The mammal may be at risk for, or suffer from, HPV infection, cervical dysplasia, and/or cervical cancer. The nucleic acids and plasmids of the invention can also be incorporated into microparticles, liposomes, ISCOMS, or any other suitable delivery vehicle as described above.
The invention further includes a plasmid having a sequence essentially identical to that of pBIOTOPEHPV (SEQ ID NO:7), or a microparticle consisting essentially of a PLGA polymeric matrix and the pBIOTOPEHPV plasmid, as well as methods of inducing an immune response in a mammal by administering either the plasmid alone, or the plasmid incorporated into such a microparticle, to the mammal.
By a xe2x80x9csubstantially pure polypeptidexe2x80x9d is meant a polypeptide which is separated from those components (proteins and other naturally-occurring organic molecules) which naturally accompany it. Typically, the polypeptide is substantially pure when it constitutes at least 60%, by weight, of the protein in the preparation. Preferably, the protein in the preparation consists of at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, of an immunogenic peptide.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The preferred methods and materials for practicing the invention are described below, although other methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present application, including definitions, will control. The materials, methods, and examples are illustrative only and not intended to be limiting.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.